Efficacy evaluation of hydrogen peroxide disinfectant based zinc oxide nanoparticles against diarrhea causing Escherichia coli in ruminant animals and broiler chickens

Different strains of Escherichia coli that exhibit genetic characteristics linked to diarrhea pose a major threat to both human and animal health. The purpose of this study was to determine the prevalence of pathogenic Escherichia coli (E. coli), the genetic linkages and routes of transmission between E. coli isolates from different animal species. The efficiency of disinfectants such as hydrogen peroxide (H2O2), Virkon®S, TH4+, nano zinc oxide (ZnO NPs), and H2O2-based zinc oxide nanoparticles (H2O2/ZnO NPs) against isolated strains of E. coli was evaluated. Using 100 fecal samples from different diarrheal species (cow n = 30, sheep n = 40, and broiler chicken n = 30) for E. coli isolation and identification using the entero-bacterial repetitive intergenic consensus (ERIC–PCR) fingerprinting technique. The E. coli properties isolated from several diarrheal species were examined for their pathogenicity in vitro. Scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HR-TEM), Fourier-transform infrared spectrum (FT-IR), X-ray diffraction (XRD), zeta potential, and particle size distribution were used for the synthesis and characterization of ZnO NPs and H2O2/ZnO NPs. The broth macro-dilution method was used to assess the effectiveness of disinfectants and disinfectant-based nanoparticles against E. coli strains. Regarding the results, the hemolytic activity and Congo red binding assays of pathogenic E. coli isolates were 55.3 and 44.7%, respectively. Eleven virulent E. coli isolates were typed into five ERIC-types (A1, A2, B1, B2, and B3) using the ERIC-PCR method. These types clustered into two main clusters (A and B) with 75% similarity. In conclusion, there was 90% similarity between the sheep samples' ERIC types A1 and A2. On the other hand, 89% of the ERIC types B1, B2, and B3 of cows and poultry samples were comparable. The H2O2/ZnO NPs composite exhibits potential antibacterial action against E. coli isolates at 0.04 mg/ml after 120 min of exposure.

environment 4 .This is a significant worldwide health concern for both people and animals.Animal output is negatively impacted by a range of E. coli infections, particularly in poultry businesses.These conditions include hemorrhagic colitis, blood poisoning diarrhea, urinary tract infections, and abdominal sepsis 5,6 .This presents a major global health risk to both humans and animals.The appropriate application of sanitary protocols is one of the primary areas of concern in cattle production systems 7 .Between 70 and 95% of cases reported globally were found to have the pathogenic strain of E. coli.In addition, E. coli strains have a substantial financial impact and are a foremost cause of illnesses in the global chicken and poultry sectors 5 .Based on its antigenic composition, the species E. coli is separated serologically into serogroups and serotypes (somatic O antigens for serogroups and flagellar or H antigens for serotypes).The third class of antigens, known as capsular or K antigens, is expressed by a large number of strains and plays a crucial role in pathogenesis 8 .
The source and path of microbial contamination have been identified using a number of molecular typing techniques [9][10][11] .Selecting a suitable approach for bacterial genotyping is contingent upon various factors, including instrument availability, cost, speed, sensitivity, strengths, and user-friendliness of databases 12,13 .Because it is simpler, quicker, and less expensive than PFGE or MLST for determining the genetic similarity of bacterial strains, a straightforward PCR-based technique called ERIC has been extensively used 14 .
ERIC as a repeat sequence is seen in bacterial genomes 15 .Several bacterial isolates, including E. coli, can have their clonal variability evaluated using these molecular biological methods 16 .Intergenic repetitive units were identified first in E. coli and Salmonella enterica serovar Typhimurium.The study of infectious disease epidemiology now incorporates molecular biology methods 17 .highlight the importance of using PCR-based genotyping methods in conjunction with serotyping for epidemiological studies of highly pathogenic E. coli strains 18 .
Several effective disinfectants became crucial to use to prevent or impede the growth of microorganisms.Furthermore, novel methods for disinfection formulas with low residual levels, like hydrogen peroxide, are needed for the present-interest products 19 .Moreover, it has been discovered that combining H 2 O 2 with other antibacterial agents increases their ability to penetrate bacterial cells and/or strengthens their oxidizing effect 20 .In addition, nano zinc oxide particles that pierce the cell wall are one of the antibacterial agents that limit the growth of bacterial infections due to oxidative stress damage 21 .Moreover, ZnO NP was discovered to have an antibacterial impact on Gram-negative bacteria, such as K. pneumoniae and E. coli 22 .
Therefore, the purpose of this work was to ascertain the prevalence rate of pathogenic E. coli in numerous diarrheic species (cows, sheep, and broiler poultry), and the virulence indicators (Congo red and hemolysis binding ability) of E. coli.As well, the genetic diversity of the most virulent strains of E. coli was characterized using ERIC-PCR.Furthermore, the degree of similarity among the isolates was determined by the development of a dendrogram, which allowed for the comparison of clusters produced by the examination of various sampling locations and evaluating the variety of potential sources of contamination.Finally, the efficiency of several disinfectants (H 2 O 2 , Virkon® S, and TH 4+ ), nano zinc oxide, and H 2 O 2 /ZnO NPs composite against the most pathogenic E. coli strains was assessed.Consequently, the current study is beneficial in preventing the incidence of diarrheal causes and their impact on animal health, as well as the breakout of pathogenic E. coli in cattle, sheep, and broiler poultry farms.

Study site and animal population
This study was carried out in a private broiler poultry, cattle, and sheep farms located in Alexandria Governorates during the period from September 2022 until October 2023.In addition to broiler chickens (n = 30), it also included ruminant animals (n = 70) at various phases of production.The cleaning and disinfection programs implemented at the farms under investigation received no particular emphasis, and the overall hygienic conditions on these farms were moderately fair.

Ethical statement
There are no experimental studies on either animals or human data in the manuscript.All methods used in this context were carried out in compliance with the rules and regulations that applied.The data gathered was all documented and statistically analyzed.

Samples collecting
Using sterile cotton swabs, 100 fresh fecal samples were directly obtained under aseptic conditions from various diarrheal species [cows (n = 30), sheep (n = 40), and broiler chickens (n = 30)].These samples were transferred on ice for 2 h until they reached the laboratory 23 .Following accurate identification, samples were sent immediately to the lab for additional microbiological analysis.

Serological typing of E. coli
Using E. coli antisera (polyvalent and monovalent O), agar slants harboring the most pathogenic and generous growth of E. coli (n = 11) were submitted for agglutination testing.Morris et al. 27 state that serological identification was used to identify E. coli.All isolates were serotyped in the Animal Health Institute's Serology Department using commercially available kits (Test Sera Enteroclon, Anti-Coli, SIFIN Berlin, Germany).

ERIC-PCR characterization of pathogenic E. coli
The QIAamp DNA Mini kit (Qiagen, Germany, GmbH) was used to extract DNA from bacterial cells of fecal samples, with certain changes made in accordance with the manufacturer's instructions.In summary, 200 µl of the bacterial suspension was treated for 10 min at 56 ° with 10 µl of proteinase K and 200 µl of lysis buffer for the degradation and digestion of proteins.200 µl of 100% ethanol was added to the lysate following incubation.After that, the sample was centrifuged and cleaned in accordance with the manufacturer's instructions.An elution buffer containing 100 µl was used to elute the nucleic acid.The oligonucleotide primers that were recorded in Table 1.For PCR amplification, primers were used in a 25 µl reaction that included 12.5 µl of Emerald Amp Max PCR Master Mix (Takara, Japan), 1 µl of each primer at a concentration of 20 pmol, 5.5 µl of water, and 5 µl of DNA template for PCR amplification.A 2720 thermal cycler from Applied Bio-system was used to carry out the reaction.The PCR products were separated by electrophoresis employing gradients of 5V/cm on a 1.5% agarose gel (Applichem, Germany, GmbH) with ethidium bromide staining in 1 × TBE buffer at room temperature.Twenty microliters of the items were put into each gel slot for the gel analysis.A Genedirex 100-3000 bp DNA ladder H3 RTU (Genedirex, Taiwan) was used to determine the fragment sizes.UV, or visible light is used by a gel documentation system (Alpha Innotech, Biometra) to stimulate fluorescent or chromogenic molecules in the gel.After the molecules produce light, an image is captured and saved by a camera.Computer software was then used to analyze the data 28 .Depending on whether each band was present or absent, the ERIC fingerprinting data was converted into a binary code.Ward's hierarchical clustering procedure and the unweighted pair group technique with arithmetic average (UPGMA) and SPSS, version 22, were used to cluster analysis and create dendrograms 29 .The online program (https:// plane tcalc.com/ 1664/) was used to calculate the number of crossing elements and the similarity index (Jaccard/Tanimoto Coefficient) between all investigated samples.

Synthesis and characterization of tested ZnO NPs and H 2 O 2 /ZnO NPs
The method of high-energy ball milling (HEBM) was used to generate ZnO NPs 31 .Subsequently, to create H 2 O 2 capping on ZnO NPs, 3% hydrogen peroxide was added to the various ZnO NP concentrations (0.02 and 0.04 mg/ mL) right before use.The mixture was then vigorously shaken on a magnetic stirrer to minimize NP agglomerations throughout the incubation times (30, 60, and 120 min).SEM (JEOL (JSM-5200), Japan), HR-TEM (a JEOL JEM 2000EX), FT-IR (VERTEX, 70), XRD (PANalytical Empyean, Sweden), zeta potential, and distribution of

Assessing antimicrobial method of disinfectants, ZnO NPs, and H 2 O 2 /ZnO NPs composite
Broth macro-dilution method was utilized to estimate the antibacterial efficacy of tested compounds.100 µl of various bacterial strains (1 × 10 −6 CFU/ml) were inoculated with 0.5% and 1% of TH 4+ disinfectant (SoGeVal, France),Virkon®S (Antec International TD, UK) at the same concentrations, hydrogen peroxide (H 2 O 2 , 6th October 3rd Industrial Area, Egypt) at a concentration of 3 and 5%, ZnO NPs (0.02 and 0.04 mg/ml), and H 2 O 2 / ZnO NPs composite (0.02 and 0.04 mg/ml) in Mueller-Hinton broth (MHB) onto a 96-well plate (Sarstedt, Numbrecht, Germany) was evaluated against thirty strains of E. coli isolates according to Li et al. 30 at different concentrations and testing times (30 min, 60 min, and 120 min).In order to generate the negative control, one microliter of broth culture was introduced to MHB without any testing materials.As a positive control, tested disinfectants and nanomaterials in MHB was conducted concurrently.A standard strain of E. coli ATCC 25,922 was applied as a quality control-positive organism.For 24 h, all of the tested materials were incubated at 37 °C.Three duplicates were used for the in-vitro experiment.In accordance with CLSI 32 recommendations, one loopful of each well was inoculated on Mueller-Hinton agar to monitor the presence or lack of microbial growth at various doses of the tested substances.

Statistical analysis
After being gathered, all of the data was entered into a Microsoft Excel spreadsheet to become available for analysis.Non-parametric tests (Chi-square test, K independent sample) using SPSS (statistical package for social sciences, version 22.0) were applied to determine the prevalence rate of pathogenic E. coli isolated from various diarrheal species, sero-grouping of some isolated strains, cluster analysis and dendrogram construction, and the bactericidal effect of testing disinfectants and nanocomposite against pathogenic E. coli, with a probability level of p ≤ 0.05.
Utilizing DNA fragments obtained through isolated E. coli bacteria from sheep, cows, and broiler chickens, the variety and quantity of bands generated from electrophoresis on gels were noted.A range of 0 to 60 bands covering 70 bp to 2161 bp was found in the ERIC-PCR band sequences.It was found that isolated strains from sheep had the greatest frequency and variety.Moreover, strains isolated from chickens showed the highest degree of similarity among DNA molecule band patterns.The isolated strains from sheep, cows, and broiler chickens' fecal samples showed prominent fragment sizes in DNA fingerprints of 1135 bp, 1184 bp, and 2161 bp, respectively; the observed bands, as illustrated in Fig. 1, ranged widely from 70 to 2161 bp.The serotyping of certain E. coli isolates obtained from various diarrheal species, as displayed in Table 4, showed that 11 (100%) of the isolated E. coli strains were typable.The most prevalent E. coli serogroup was O26:K60 (3), which was followed by O44:K74(2), O124:K72(2), O25:K11(2), O118: K-(1), and O78: K-(1).www.nature.com/scientificreports/ In the present investigation, eleven virulent E. coli isolates were typed into ERIC-types using ERIC-PCR profiles.Using a 75% similarity limit, dendogram analysis separated them into two large clusters, A and B. Cluster A is separated into two groups, A1 and A2, containing five isolates that are sheep-related.The distribution of E. coli isolate numbers in group A1 is "3, 4, and 2", while in group A2 it is "1 and 5", respectively.Ninety percent of these two groups were comparable.With 6 isolates (cows (n = 4), which included E. coli isolates number "8, 9, 7, 10'') and broiler chickens (n = 2), which contained E. coli isolates number "10 and 11"), Cluster B was classified into groups (B1, B2, and B3).There was an 89% similarity between these three groups.Furthermore, for B1, B2, Table 3. Pathogenicity determinants of E. coli isolates from different diarrheic species.Pathogenicity determinants of E. coli isolates among diarrheic species was significantly different at (χ 2 ) = 114, P ≤ 0.05.*% according to total number of E. coli isolates recovered from each species.** % according to total number of E. coli isolates recovered from all species.and B3, the similarity within each group was 96%, 94%, and 92%, respectively (Fig. 2).All E. coli isolates had an identity range of 0.17 to 1, but samples from sheep, cows, and broiler chickens had ranges of 0.67-1, 0.22-0.6,and 0.67-0.17,respectively (Fig. 3).The antimicrobial sensitivity profile of testing disinfectants (TH 4+ , Virkon®S, and H 2 O 2 ), ZnO NPs, and H 2 O 2 / ZnO NPs composite against pathogenic E. coli in Table 5 clarified that all isolated pathogenic E. coli and the control-positive strain (E. coli ATCC 25,922) were found to be completely sensitive to TH 4+ at a concentration of 1:100 ml after 120 min of exposure time at P ≤ 0.05.In addition, the sensitivity of E. coli did not exceed 70% at the least concentration (1:200 ml) after 120 min of contact time.Conversely, Virkon®S disinfectant proved to be 100% effective against E. coli and E. coli ATCC 25,922 at a dosage of 1:100 ml after 120 min of contact time at P < 0.05.In contrast, the sensitivity testing of E. coli isolates to H 2 O 2 was significantly low at different contact times and did not exceed 50% at 5% concentration after time exposure (120 min) at P ≤ 0.01 compared to the lowest concentration of 3%.Oppositely, nano zinc oxide was verified to have a lethal effect (100%) on E. coli  www.nature.com/scientificreports/and a control positive stain at 0.04 mg/ml after 120 min.It's interesting to note that employing nano zinc oxide increases hydrogen peroxide's ability to penetrate bacterial cells.In comparison to other doses, it was discovered that hydrogen peroxide loaded on ZnO NPs was highly effective (100%) against all E. coli isolates and the control positive one at 0.04 mg/ml after 120 min of exposure compared to other concentrations.SEM microscopy of ZnO NPs, as shown in Fig. 4a.It emerged as uniform, spherical particles loaded on top of one another.After loading, H 2 O 2 /ZnO NPs (Fig. 4b) seemed to be a lot of elongated particles in shape.The morphological feature of nano zinc oxide (Fig. 5a) was revealed to be hexagonal, and the diameter of the NPs ranged from 75.08 to 100.58 nm (Fig. 5b), according to TEM microscopy.Additionally, TEM micrographs of H 2 O 2 /ZnO NPs revealed that the nanoparticles' shape had changed to a pentagonal form (Fig. 5c), and their diameter ranged from 5.48 to 34.6 nm (Fig. 5d).On the other hand, FTIR spectra of the hydrogen peroxide, nano zinc oxide, and H 2 O 2 loaded on ZnO NPs, as shown in (Fig. 6) clarified that nano zinc oxide exhibited strong absorption peaks at 3435, 2372, 1637, 1044, 723, and 535 cm −1 (Fig. 6a).H 2 O 2 revealed a wide range of absorption peaks linked to the absorption of hydroxyl groups (O-H).Moreover, characteristic peaks were observed at 3265, 2353, 2122, 1636, 1387, 1210, and 600 cm −1 , respectively (Fig. 6b).Additionally, the composite H 2 O 2 /ZnO NPs (Fig. 6c) demonstrated the strongest peak migrated to 3270 and 2350 cm −1 , in addition to characteristic stretching mode vibration peaks at 1346 and 615 cm −1 , confirming the interaction between the tested disinfectant (H 2 O 2 ) and nano zinc oxide.The structural properties of ZnO NPs, and H 2 O 2 /ZnO NPs composite were examined through XRD diffraction, as displayed in Fig. 7.The XRD pattern of ZnO NPs exhibited high crystallinity, where the presence of 100, 002, 101, and 110 planes matched the hexagonal crystal structure of nano zinc oxide.Besides, the intensity of peaks decreased in H 2 O 2 /ZnO NPs, exhibiting a decrease in the crystallinity of the composite.Oppositely, the stability and nanoparticle charge were measured using zeta potential (Fig. 8) based  on their electrophoretic mobility.H 2 O 2 /ZnO NPs composite (Fig. 8a) had a negative charge of − 0.12 mV, and the hydrodynamic diameter of the particle size was 2625 nm (Fig. 8b).

Discussion
Globally, enterotoxigenic E. coli (ETEC) bacteria are acknowledged as a significant contributor to the general issue of diarrhea 33 .Cattle are a natural reservoir for E. coli in livestock; where the bacteria are always carried in their feces and can infect anywhere from 1 to 50% of healthy cows 34 .Preventing an E. coli outbreak can be achieved by regularly monitoring of animals and enforcing strict hygiene measures during every stage of production and carried out at every stage of the supply chain, from farms to the employees who handle the animals.Rural farmers should look into the details and become more knowledgeable about different diets, their components, and the application of antibacterial agents.In emerging nations, epidemiological and pathogenic characteristics linked to the E. coli strain require more examination.Regular examinations of this pathogen are also necessary, particularly in urban and rural areas 35,36 .
Escherichia coli is one of the model organisms that is most thoroughly investigated 37,38 .ERIC-PCR is one of many techniques used to determine bacterial transmission.Various studies have employed it for a variety of bacterial isolates, including E. coli, Salmonella spp., Pseudomonas aeruginosa, and Streptococcus [39][40][41] .The current investigation found that 38 isolates out of 100 samples from various diarrheic species (cows, sheep, and broiler chickens) contained E. coli, with a total prevalence of 38%.In contrast, the prevalence rates in each of the diarrheal species were 33.3%, 37.5%, and 43.3%, respectively, as shown in Table 2.This finding was in line with those of previous studies, which confirm that E. coli is one of the major bacteria that cause diarrhea in sheep, broiler chickens, and cows.Moreover, Fouad et al. 42 and Algammal et al. 43 reported that the E. coli prevalence in diarrheic calves was 37.4% and 28.8%, respectively.According to Khalil et al. 44 , 30.2% of the 16 out of 53 sheep rectal swab samples with diarrheal symptoms had positive E. coli isolates.Meanwhile, Hafez 45 found that the E. coli prevalence was high at 69.7% in diarrheal sheep.Oppositely, from diarrheal broiler, E. coli isolates were found in 40% and 20% of the governorates of El-Fayoum and Giza, respectively, according to EL-Demerdash et al. 46 .A number of variables, including the raising system, the surroundings, the age of the birds, their immunity, and their stage of production, may be responsible for this variance.The pathogenicity of the E. coli strains-their capacity to cause hemolysis and bind to Congo red-was assessed in the existing study.Strains of E. coli exhibited both beta-and -alpha hemolysis.Since hemolysis was shown to induce cell membrane damage, it was employed as a phenotypic marker for the pathogenicity factor of E. coli.Additionally, 55.3% of the E. coli isolates found in diarrheal sheep, broiler chickens, and cows were betahemolytic.These almost match the findings of Abd El-Wahed 47 , who reported that 66.7% of the tested isolates of E. coli were hemolytic.Furthermore, 44.7% of the entire E. coli strains that were recovered from various diarrheal species displayed CR positivity, albeit to varying degrees of redness.Fouad et al. 42 discovered that, to varying degrees, 60.6% of the E. coli isolates under investigation tested positive for CR.Cong red is a straightforward dye that can be added to agar media.Quinn et al. 48reported that dye uptake has been shown to be a virulence marker to differentiate between invasive and noninvasive isolates.44.7% of the total E. coli strain recovered from diarrheal sheep, cows, and broiler chickens in the current investigation demonstrated CR positivity, but to varying degrees of red color (40%, 46.7%, and 46.2%, respectively).These findings were not as promising as those of Fouad et al. 42 , who discovered that 60.6% of the tested E. coli isolates had varying degrees of CR positivity.The most popular epidemiological marker for classifying pathogenic E. coli is thought to be serotyping.Particularly when it comes to E. coli that causes diarrhea, some serotypes are known to be closely linked to pathotypes.In order to better understand E. coli epidemiology and control the bacteria that cause diarrhea and non-intestinal illnesses, it is more beneficial to analyze the incidence of different E. coli serotypes and their distribution patterns across different geographic locations.Eleven E. coli isolates were identified using serological analysis.All strains (100%) could be typed.The most prevalent serogroup was O26:K60 (3), followed by O44:K74(2), O124:K72(2), O25:K11(2), O118:K-(1) and O27:K-(1).When E. coli strains were obtained from sheep, serogroups O26:K60 and O44:K74  www.nature.com/scientificreports/were found, whereas isolates from cows had O124:K72 and O25:K11, and isolates from chickens had O118:K-and O78:K-.The E. coli serogrouping is shown in Table 4.The E. coli strains were identified serologically as O157:H7 (n = 4; two isolated from calves and two from goat kids), O125 (n = 3; two isolated from calves and one from lambs), and O44 (n = 3; two isolated from goat kids and one from lambs), according to Abd EL-Tawab et al. 49 Meanwhile, Algammal et al. 43 identified seven serogroups (O26, O45, O91, O111, O119, O125, and O128) by serotyping the E. coli strains from calf diarrhea.Furthermore, Wilczy´nski et al. 50and El-Mongy et al. 51 reported that serotype O78 was the most common serotype among E. coli isolates from all varieties of chickens.ERIC-PCR profiles in our study allowed us to classify virulent E. coli isolates into ERIC-types.Using a 75% similarity limit, dendogram analysis separated them into two large clusters, A and B. Cluster A was split up into A1 and A2 groups.Ninety percent of isolated E. coli strains from the two groups A1 and A2 of diarrheagenic sheep were comparable.Cluster B was split up into groups (B1, B2, and B3).There was an 89% similarity between these three groups.Furthermore, for B1, B2, and B3, the corresponding levels of similarity within each group were 96%, 94%, and 92%.The range of identities for all E. coli isolates was 0.17 to 1, with corresponding ranges for sheep, cows, and poultry samples (0.67 to 1), (0.22 to 0.6), and (0.67 to 0.17) as shown in Figs. 2 and 3. Sekhar et al. 18 revealed that ERIC-PCR was demonstrated to be a quick, sharp, and cost-effective fingerprint approach for successful discrimination of E. coli isolates based on their genotype.There may be complex transmission of E. coli from broiler chickens to cows and the environment, and vice versa, as evidenced by the high DNA fingerprint relatedness shared by several strains of the bacteria from different animals and broiler chickens.Our findings revealed crucial information on the genetic and epidemiological traits of E. coli and emphasized the need for stronger biocontainment measures in order to lower the occurrence and effects of the bacteria in animal and poultry husbandry.
The susceptibility pattern of pathogenic E. coli to three different disinfectant compounds (TH 4+ , Virkon®S, and H 2 O 2 ) was found to be as follows: after 120 min of exposure time at a concentration of 1:100 ml, all testing bacterial strains of E. coli were completely sensitive to testing disinfectants TH 4+ and Virkon®S, while the effectiveness of H 2 O 2 on E. coli isolates was not greater than 50% at 5% concentration after 120 min of exposure.These results were consistent with those of Fawzia et al. 52 , who discovered that E. coli isolates were susceptible (86.7%) to Virkon®S (1%) and TH 4+ (0.2%) using the disc diffusion method.Gehan et al. 53 found that the synergy of glutaraldehyde and QAC makes TH 4+ the most potent disinfectant.Additionally, glutaraldehydebased disinfectants showed a high degree of sensitivity against both S. aureus and E. coli 54 .Conversely, Rutala and Weber 55 indicated that H 2 O 2 at a concentration of 7.5% was the most effective disinfectant among the oxidizing agents.After five minutes of exposure, R´ıos-Castillo et al. 20 discovered that H 2 O 2 integrated with cationic polymers at the same concentration was very effective.According to Lineback et al. 56 , H 2 O 2 disinfection outperformed quaternary ammonium compounds (QACs) in its ability to destroy P. aeruginosa and S. aureus biofilms.In this study, following 120 min of exposure, nano zinc oxide was shown to have bactericidal effects on E. coli at the maximum dose (0.04 mg/ml).Thus, these results allowed us to investigate the possibility of employing nano zinc oxide to increase hydrogen peroxide's ability to penetrate bacterial cells.It's interesting to note that, in contrast to other concentrations, hydrogen peroxide loaded on ZnO NPs was shown to have a deadly effect against all E. coli isolates (100%) at the same concentration and exposure period (0.04 mg/ml and 120 min), whereas the diameter of the NPs ranged from 75.08 to 100.58 nm.Moreover, ZnO NPs have the potential to be antimicrobial effective (average size = 30 nm), causing bacterial cell death by disrupting the integrity of the cell wall 57 .Furthermore, Siddiqi et al. 58 found that at 125 μg/ml, micro zinc oxide particles had a high level of efficiency against S. aureus and E. coli.Additionally, the average size of NPs ranged from 5.48 to 34.6 nm.Abdelghany et al. 59 observed that ZnO NPs had antibacterial activity against various species of bacteria such as S. aureus, E. coli, and K. pneumonia with inhibition zones (23.83 ± 0.29, 28.33 ± 0.58, and 23.83 ± 1.04, respectively).ZnO NPs had a biocidal effect by accumulating nanoparticles in the cytoplasm and/or outer bacterial cell wall, which released Zn 2+ and damaged membrane proteins, killing the microbial cell 60,61 .With regards to the SEM image, it displayed randomly distributed ZnO NPs with aggregated particles.Furthermore, peaks at 602.64 cm −1 in the FT-IR spectra of the biosynthesized ZnO NPs are linked to the ZnO stretching vibration mode.ZnO NPs' XRD pattern showed a monoclinic structure.The hexagonal phase crystals of zinc oxide were confirmed by the usual diffraction peaks detected at 2θ = 31.72°(100), 34.47 (002), 36.25 (101), 47.71 (102), 56.59 (110), 62.98 (103), and 67.78 (112).The average particle size of ZnO NPs ranged from 12.4 to 18.9 nm 62 .

Conclusion
The prevalence rate of pathogenic E. coli was significantly higher in poultry feces (43.3%) than that of sheep and cows.All E. coli isolates from various diarrheagenic animals were recognized using ERIC-PCR, whereas the identities of sheep, cows, and broiler chickens varied from 0.67 to 1.0, 0.22 to 0.6, and 0.67 to 0.17, respectively.Testing E. coli strains were particularly susceptible to the disinfectants TH 4+ and Virkon®S after 120 min of exposure at a dosage of 1:100 ml.The efficacy of H 2 O 2 against E. coli was not greater than 50% at 5% concentration during any testing contact period.It's interesting to note that the H 2 O 2 /ZnO NPs composite exhibits possible antibacterial action against E. coli isolates at 0.04 mg/ml after 120 min of exposure.The promising composite proved its stability, and based on their electrophoretic mobility, it had a negative charge of − 0.12 mV, and the hydrodynamic diameter of the particle size was 2625 nm.

Figure 3 .
Figure 3. Genetic Similarity index of eleven virulent E. coli isolates.

Figure 5 .
Figure 5. Transmission electron microscopy of ZnO NPs (a-b) clarified the hexagonal shape of zin oxide nanoparticles (a) and the diameter of NPs was ranged between 75.08 to 100.58 nm (b).Moreover, H 2 O 2 /ZnO NPs Micrographs exhibited the alteration in NPs shape to pentagonal (c) and the size of NPs in diameter was ranged from 5.48 to 34.6 nm (d).

Table 1 .
Primer sequence, target gene, amplicon sizes and cycling conditions.Malvern Instruments Ltd., Worcestershire, UK) were used to characterize both nano zinc oxide and H 2 O 2 /ZnO NPs.In the Central Lab of the Agriculture Faculty at Cairo University, Egypt, HR-TEM, and SEM micrographs were done.While at Beni-Suef University's Faculty of Postgraduate Studies of Advanced Science, the nanocomposite's FTIR spectra, XRD, particle size distribution, and zeta potential were achieved.

Table 2 .
Prevalence rate of pathogenic E. coli isolated from different diarrheic species.The chi-square association of prevalence rate of pathogenic E. coli isolates in collected samples is statistically significant at (χ 2 ) = 94, P ≤ 0.05.

Table 5 .
Antimicrobial efficiency of testing disinfectants and nanomaterials against pathogenic E. coli.